1. Field of the Invention
The present invention relates generally to the delivery of nucleic acid molecules into cells and, more specifically, to compositions and methods for the high efficiency delivery of nucleic acid molecules into cells.
2. Background Information
Methods that allow the introduction and expression of foreign or exogenous nucleic acid molecules into cells in culture are useful for manipulating the function and expression of various genes, as well as for efficiently expressing a desired protein. In addition, the ability to transfer genes to human cells provides the means to treat a wide range of genetic and acquired diseases, including cystic fibrosis, sickle cell anemia, and AIDS. Unfortunately, methods for introducing a gene of interest into a cell have provided relatively low transfection efficiencies or have been successful only in cultured cell lines.
Transfection methods can be used to replace a defective gene or to correct an error in an existing one. Replacement therapy entails inserting a gene into a cell in order to synthesize a gene product that is not being produced or is being synthesized in inadequate amounts. Corrective therapy, on the other hand, attempts to correct an error in a gene by providing conditions for a recombinational event that replaces all or part of the defective gene with the correct DNA sequence.
Various transfection methods have been developed for eukaryotic cells, particularly mammalian cells. Some transfection methods use calcium phosphate or DEAE-dextran as a carrier to promote the uptake of an exogenous nucleic acid molecule. Other methods use xe2x80x9clipofectionxe2x80x9d techniques, which incorporate the use of synthetic anionic or cationic lipids to effect the transfection. Osmotic shock, treatment of the cells with liposomal inhibitors, and high voltage electric pulses, which create pores in cell membranes, also have been used in attempts to enhance transfection efficiencies. However, the efficiencies obtained by these methods are relatively low, ranging from 0.001% to 1%, depending on the recipient cell line.
In further efforts to increase the efficiency of introducing nucleic acid molecules into cells, viral vectors have been used. For example, when retroviral vectors are used, the introduced DNA replaces some of the retroviral genes required for the production of viral structural proteins while the viral sequences directing integration into the cellular DNA remain intact. However, the use of retroviral vectors is limited to dividing cells. Adenovirus vectors also have been used with fairly high efficiency, but they only infect specific cell types. In addition, the use of viral vectors is limited by the induction of an immune response against the viral components of the vectors.
Other methods of introducing a nucleic acid molecule into a cell include complexing the nucleic acid molecule with proteins that bind to specific receptors expressed by the target cells, or by incorporating the nucleic acid molecule into liposomes, which fuse with the target cell membrane. Gene transfection of mammalian cells using cationic liposomes, for example, has achieved an efficiency of up to 15% and transferrin-poly-L-lysine mediated transfection has yielded 7% to 8% transfection efficiency. A transfection efficiency of about 90% has been achieved in a cultured cell line using a combination of transferrin and cationic liposomes.
While these approaches represent an improvement over prior techniques, a need exists for compositions and methods for introducing a nucleic acid molecule into any target cell, particularly primary mammalian cells, with efficiencies of about 50% or greater. The present invention satisfies this need and provides related advantages as well.
The present invention provides a composition for introducing a nucleic acid molecule into a target cell, comprising a liposome, ligand and polymeric scaffold, wherein the ligand can bind to a receptor on the target cell. The liposome can be cationic. The ligand can be attached to the polymeric scaffold. The polymeric scaffold can be positively charged. Thus, for example, the invention provides a composition comprising a cationic liposome, and transferrin, wherein the transferrin is attached to poly-L-lysine.
The invention also provides a method for introducing a nucleic acid molecule into a target cell by contacting the target cell with the nucleic acid molecule, a liposome, a ligand and a polymeric scaffold, wherein the ligand binds to a cell surface receptor on the target cell. The ligand can be attached to the polymeric scaffold. The polymeric scaffold can be positively charged. Thus, for example, the invention provides a method for introducing a nucleic acid molecule into primary mammalian cells, wherein at least about 50% of the transfected cells contain the introduced nucleic acid molecule. A method of the invention further comprises permeabilizing the target cells to facilitate uptake of the nucleic acid molecule.